Influenza viruses in the form of a flu epidemic or a pandemic occurring every year threaten human health. As the most efficient method for preventing this, a vaccine is used. Vaccines are divided into two types, an inactivated vaccine using a surface protein of a virus as an antigen, and a live vaccine with an attenuated virus. In live vaccine preparation methods developed up to now, a cold adapted attenuated live vaccine has been mainly used as a prevention vaccine (Watanabe, S. et al., Influenza A Virus Lacking M2 Protein as a Live Attenuated Vaccine. J Virol 83, 5947-5950 (2009); Steel, J. et al., Live Attenuated Influenza Viruses Containing NS1 Truncations as Vaccine Candidates against H5N1 Highly Pathogenic Avian Influenza. J Virol 83, 1742-1753 (2009); Perez, J. T. et al., MicroRNA-mediated species-specific attenuation of influenza A virus. Nat Biotech 27, 572-576 (2009); Stech, J. et al., A new approach to an influenza live vaccine: modification of the cleavage site of hemagglutinin. Nat Med 11, 683-689 (2005)). However, live vaccines have safety problems, and thus their use for infants, old people, or some people with a reduced immunity level is restricted (Cox, R. J. et al., Influenza Virus: Immunity and Vaccination Strategies. Comparison of the Immune Response to Inactivated and Live, Attenuated Influenza Vaccines. Scandinavian Journal of Immunology 59, 1-15 (2004)).
As a method for improving the safety of a live vaccine, there has been recently suggested a method of employing two or more attenuation methods in one kind of live vaccine. As a conventional technology, there is a method for introducing a cold-adaptation character into a virus, thereby resulting in propagation inhibition at a regular human body temperature of 36 to 37° C. (Monto, A. S. et al., Evaluation of an attenuated, cold-recombinant influenza B virus vaccine. J Infect Dis 145, 57-64 (1982); Lee, K.-H. et al., Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus. Vaccine 24, 1966-1974 (2006); Belshe, R. B. et al., Current status of live attenuated influenza virus vaccine in the US. Virus Research 103, 177-185 (2004); Seo, S.-U. et al., Development and characterization of a live attenuated influenza B virus vaccine candidate. Vaccine 26, 874-881 (2008)). As another conventional technology, there is a method for attenuating a virus by removing or modifying a nonstructural protein 1 (NS1) from among influenza virus proteins. Also, there has been developed a method for attenuating a virus by removing M2 ion channel protein, and modifying hemagglutinin (HA) and a protein cleavage site, Further, there has been recently developed a method for reducing gene replication efficiency of a virus within a cell by using a gene silencing mechanism of miRNA.
Meanwhile, virus attenuation often even causes the destruction of propagation capability of a virus in a fertilized egg. Accordingly, in preparation of an attenuated live vaccine, sometimes, it is required to change a preparation system from a fertilized egg to a cell line (such as a MDCK cell line or a Vero cell line). Also, in some cases, an expensive enzyme required for virus propagation has to be added to a cell culture fluid.
Meanwhile, the inventors of the present invention developed X-31 ca as a donor strain of a cold-adapted attenuated live vaccine, which has a strong immunogenicity and a high propagation capability at a replication-competent temperature, and has a high attenuation at a non-replication-competent temperature (Lee, K.-H. et al., Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus. Vaccine 24, 1966-1974 (2006)). The X-31 ca virus can be replicated in a lung and an upper airway of a mouse through infection (104 PFU) in spite of its high attenuation character. The infection with a live vaccine in a host cell is apparently directly related to the immunogenicity. However, a long-term survival of a virus in an infected host cell may cause a toxic problem such as spontaneous genetic variation and the virus' reassortment together with another human virus (Cox, R. J., Brokstad, K. A. & Ogra, P. Influenza Virus: Immunity and Vaccination Strategies. Comparison of the Immune Response to Inactivated and Live, Attenuated Influenza Vaccines. Scandinavian Journal of Immunology 59, 1-15 (2004)).
Accordingly, in order to eliminate a live vaccine's latent risk raised from the conventional technology and previous research, the inventors of the present invention have tried to develop an attenuation method which can more quickly remove a virus from a virus-infected cell, and can improve stability of a live vaccine without damage to immunogenicity and high productivity, and then thus have completed this invention.
The above information disclosed in this Background section is only for enhancement of understanding of the background of the invention and therefore it may contain information that does not form the prior art that is already known in this country to a person of ordinary skill in the art.